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Reaction Amplification (reaction + amplification)
Kinds of Reaction Amplification Selected AbstractsPatterns of bacterial diversity across a range of Antarctic terrestrial habitatsENVIRONMENTAL MICROBIOLOGY, Issue 11 2007Etienne Yergeau Summary Although soil-borne bacteria represent the world's greatest source of biological diversity, it is not well understood whether extreme environmental conditions, such as those found in Antarctic habitats, result in reduced soil-borne microbial diversity. To address this issue, patterns of bacterial diversity were studied in soils sampled along a > 3200 km southern polar transect spanning a gradient of increased climate severity over 27° of latitude. Vegetated and fell-field plots were sampled at the Falkland (51°S), South Georgia (54°S), Signy (60°S) and Anchorage Islands (67°S), while bare frost-sorted soil polygons were examined at Fossil Bluff (71°S), Mars Oasis (72°S), Coal Nunatak (72°S) and the Ellsworth Mountains (78°S). Bacterial 16S rRNA gene sequences were recovered subsequent to direct DNA extraction from soil, polymerase chain reaction amplification and cloning. Although bacterial diversity was observed to decline with increased latitude, habitat-specific patterns appeared to also be important. Namely, a negative relationship was found between bacterial diversity and latitude for fell-field soils, but no such pattern was observed for vegetated sites. The Mars Oasis site, previously identified as a biodiversity hotspot within this region, proved exceptional within the study transect, with unusually high bacterial diversity. In independent analyses, geographical distance and vegetation cover were found to significantly influence bacterial community composition. These results provide insight into the factors shaping the composition of bacterial communities in Antarctic terrestrial habitats and support the notion that bacterial diversity declines with increased climatic severity. [source] Prokaryotic diversity and metabolically active microbial populations in sediments from an active mud volcano in the Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 10 2006Robert J. Martinez Summary In this study, ribosomes and genomic DNA were extracted from three sediment depths (0,2, 6,8 and 10,12 cm) to determine the vertical changes in the microbial community composition and identify metabolically active microbial populations in sediments obtained from an active seafloor mud volcano site in the northern Gulf of Mexico. Domain-specific Bacteria and Archaea 16S polymerase chain reaction primers were used to amplify 16S rDNA gene sequences from extracted DNA. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from each sediment depth that had been subjected to reverse transcription polymerase chain reaction amplification. Twelve different 16S clone libraries, representing the three sediment depths, were constructed and a total of 154 rDNA (DNA-derived) and 142 crDNA (RNA-derived) Bacteria clones and 134 rDNA and 146 crDNA Archaea clones obtained. Analyses of the 576 clones revealed distinct differences in the composition and patterns of metabolically active microbial phylotypes relative to sediment depth. For example, ,- Proteobacteria rDNA clones dominated the 0,2 cm clone library whereas ,-Proteobacteria dominated the 0,2 cm crDNA library suggesting , to be among the most active in situ populations detected at 0,2 cm. Some microbial lineages, although detected at a frequency as high as 9% or greater in the total DNA library (i.e. Actinobacteria, ,- Proteobacteria), were markedly absent from the RNA-derived libraries suggesting a lack of in situ activity at any depth in the mud volcano sediments. This study is one of the first to report the composition of the microbial assemblages and physiologically active members of archaeal and bacterial populations extant in a Gulf of Mexico submarine mud volcano. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Isolation and properties of methanesulfonate-degrading Afipia felis from Antarctica and comparison with other strains of A. felisENVIRONMENTAL MICROBIOLOGY, Issue 1 2005S. Azra Moosvi Summary Three novel strains of methylotrophic Afipia felis were isolated from several locations on Signy Island, Antarctica, and a fourth from estuary sediment from the River Douro, Portugal. They were identified as strains of the ,-2 proteobacterium A. felis by 16S rRNA gene sequence, analysis., Two, strains, tested, were, shown to contain the fdxA gene, diagnostic for A. felis. All strains grew with methanesulfonate (and two strains with dimethylsulfone) as sole carbon substrate. Growth on methanesulfonate required methanesulfonate monooxygenase (MSAMO), using NADH as the reductant and stimulated by reduced flavin nucleotides and Fe(II). Polymerase chain reaction amplification of DNA from an Antarctic strain showed a typical msmA gene for the ,-hydroxylase of MSAMO, and both Antarctic and Portuguese strains contained mxaF, the methanol dehydrogenase large subunit gene. This is the first report of methanesulfonate-degrading bacteria from the Antarctic and of methylotrophy in Afipia, and the first description of any bacterium able to use both methanesulfonate and dimethylsulfone. In contrast, the type strain of A. felis DSM 7326T was not methylotrophic, but grew in defined mineral medium with a wide range of single simple organic substrates. Free-living Afipia strains occurring widely in the natural environment may be significant as methylotrophs, degrading C1 -sulfur compounds, including the recalcitrant organosulfur compound methanesulfonate. [source] ,-Globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in LebanonEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2003A. Inati Abstract: Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the , -globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the , -globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the ,,G,,A,,,,,,,, -globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF < 5%, Group B: HbF between 5 and 15%, and Group C: HbF > 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon. [source] Viral meningoencephalitis: a review of diagnostic methods and guidelines for managementEUROPEAN JOURNAL OF NEUROLOGY, Issue 8 2010I. Steiner Background:, Viral encephalitis is a medical emergency. The prognosis depends mainly on the pathogen and host immunologic state. Correct immediate diagnosis and introduction of symptomatic and specific therapy has a dramatic influence upon survival and reduces the extent of permanent brain injury. Methods:, We searched the literature from 1966 to 2009. Recommendations were reached by consensus. Where there was lack of evidence but consensus was clear, we have stated our opinion as good practice points. Recommendations:, Diagnosis should be based on medical history and examination followed by CSF analysis for protein and glucose levels, cellular analysis, and identification of the pathogen by polymerase chain reaction amplification (recommendation level A) and serology (level B). Neuroimaging, preferably by MRI, is essential (level B). Lumbar puncture can follow neuroimaging when immediately available, but if this cannot be performed immediately, LP should be delayed only under unusual circumstances. Brain biopsy should be reserved only for unusual and diagnostically difficult cases. Patients must be hospitalized with easy access to intensive care units. Specific, evidence-based, antiviral therapy, acyclovir, is available for herpes encephalitis (level A) and may also be effective for varicella-zoster virus encephalitis. Ganciclovir and foscarnet can be given to treat cytomegalovirus encephalitis, and pleconaril for enterovirus encephalitis (IV class evidence). Corticosteroids as an adjunct treatment for acute viral encephalitis are not generally considered to be effective, and their use is controversial, but this important issue is currently being evaluated in a large clinical trial. Surgical decompression is indicated for impending uncal herniation or increased intracranial pressure refractory to medical management. [source] The Effects of Cyclooxygenase2,ProstaglandinE2 Pathway on Helicobacter pylori -Induced Urokinase-Type Plasminogen Activator System in the Gastric Cancer CellsHELICOBACTER, Issue 3 2008Junichi Iwamoto Abstract Background:, Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the destruction of the extracellular matrix and basement membrane. The induction of uPA and uPAR in the gastric cancer cells with H. pylori has been demonstrated previously. The involvement of COX-2-PGE2 pathway in the uPA system (uPA and uPAR) expression is unclear. Methods:, Gastric cancer cells (MKN45) were co-cultured with H. pylori standard strain (NCTC11637). The specific inductions of uPA and uPAR mRNA were examined by reverse transcription-polymerase chain reaction amplification. The secreted uPA antigen was measured by ELISA. To evaluate the involvement of COX-2 and PGE2 pathway in H. pylori -induced uPA and uPAR expressions, we examined the effects of COX-2 inhibitor and PGE2 receptor antagonist on H. pylori -induced uPA and uPAR expression in the gastric cancer cells. Results:, The expressions of both uPA and uPAR mRNAs in the gastric cancer cells increased obviously (12-fold and 3-fold, respectively) with H. pylori stimulation. The amount of uPA antigen into the culture medium increased dramatically with H. pylori stimulation. The COX-2 expression level in the gastric cancer cells increased remarkably with H. pylori stimulation. H. pylori -induced uPA and uPAR expression levels were suppressed with COX2 inhibitor treatment. The amount of PGE2 antigen into the culture medium increased dramatically 24 hours after H. pylori stimulation. The gastric cancer cells expressed EP2 and EP4 subtypes. EP2 receptor antagonist suppressed the H. pylori -induced uPA and uPAR expressions in the gastric cancer cells. Conclusions:, Our results indicated that COX2-PGE2 pathway may be involved in H. pylori- associated uPA and uPAR induction, and that COX-2 inhibitor or EP2 receptor antagonist may inhibit angiogenesis and tumor invasion via suppression of the uPA system. [source] Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis,HEPATOLOGY, Issue 6 2010Krishna Banaudha Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. Conclusion: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease. Hepatology 2010;51:1922,1932 [source] Concordant loss of heterozygosity of DNA repair gene, hOGG1, in melanoma in situ and atypical melanocytic hyperplasiaJOURNAL OF CUTANEOUS PATHOLOGY, Issue 6 2008Shayesteh Pashaei Background:, One major risk factor for cutaneous melanoma is chronic sun-exposure and oxidative stress. Among various oxidative DNA damages, 8-oxoquanine is the most abundant and is potentially mutagenic if not sufficiently repaired. The human 8-oxoquanine DNA glycosylase 1 (hOGG1) gene specifically repairs 8-oxoguanine, and this gene shows frequent loss of heterozygosity (LOH) in human tumors. In this study, we investigate whether hOGG1 LOH occurs in melanoma in situ (MIS) and adjacent atypical melanocytic hyperplasia (AMH). Methods:, Twelve skin biopsies with MIS and adjacent AMH were included. DNA samples derived from manual microdissection of tissues were subjected to polymerase chain reaction amplification using three fluorescent-labeled microsatellite makers, followed by fragment analysis. Results:, Five of 12 cases were informative for both telomeric (3S1297) and centromeric (3S1289 or 3S1300) markers, bordering the hOGG1 locus. Among them, four (80%) MIS and three (60%) AMH showed hOGG1 LOH at both markers. Conclusions:, These results shows that LOH at hOGG1 gene is associated with MIS and AMH and suggest that the hOGG1 gene may play a role in melanocytic tumor progression. [source] Atypical growth of Renibacterium salmoninarum in subclinical infectionsJOURNAL OF FISH DISEASES, Issue 1 2006V Hirvelä-Koski Abstract Two growth types of Renibacterium salmoninarum were isolated from subclinically infected rainbow trout, one producing the smooth colonies typical of R. salmoninarum and the other forming a thin film on the surface of the agar with no separate colonies. The atypical growth was present on kidney disease medium agar in primary cultures of the kidney but not on selective kidney disease medium (SKDM). Fluorescent antibody staining of the fresh isolate and polymerase chain reaction amplification were the most reliable techniques to identify the atypical growth of R. salmoninarum. The condition was reversible, with growth reverting from atypical to the smooth colony form in experimentally infected rainbow trout and under laboratory conditions. There was no mortality, or any clinical signs of bacterial kidney disease (BKD) in the fish challenged with the atypical growth, although small numbers of smooth colonies of R. salmoninarum were isolated from 8% of these fish. The atypical growth reported here may explain some of the failures of culture, when SKDM agar alone is used for the detection of BKD in subclinically infected fish. [source] DETERMINATION OF HISTAMINE AND BACTERIAL ISOLATION IN MARLIN FILLETS (MAKAIRA NIGRICANS) IMPLICATED IN A FOODBORNE POISONINGJOURNAL OF FOOD SAFETY, Issue 3 2010H.C. CHEN ABSTRACT An incident of foodborne poisoning causing illness in seven victims due to ingestion of marlin fillets occurred in August, 2008, in Kaohsiung City, southern Taiwan. The two suspected marlin samples contained 47.8 and 43.5 mg/100 g of histamine, which is greater than the 5.0 mg/100 g allowable limit suggested by the U.S. Food and Drug Administration. Given the allergy-like symptoms of the victims and the high histamine content in the suspected marlin samples, this foodborne poisoning was strongly suspected to be due to histamine intoxication. Two histamine-producing bacterial strains capable of producing 3.10 ppm and 4.20 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l -histidine (TSBH) were identified as Bacillus subtilis by 16S rDNA sequencing with polymerase chain reaction amplification. However, major histamine-forming bacteria might have been killed during the preparation of fillets before serving and these two B. subtilis isolates might not be the main contributors to histamine accumulation in suspected fillets. PRACTICAL APPLICATIONS Based on the finding that high contents of histamine (>40 mg/100 g) were detected in the suspected marlin samples, we speculate the temperature abuse of the fillets before cooking contributed to the presence of high histamine levels in marlin fillets and resulted in foodborne poisoning. Although two histamine-producing Bacillus subtilis strains were isolated from suspected fish samples, both might not to be the main contributors to histamine accumulation because of low histamine production. These results re-emphasize proper handling temperature for seafoods and offer an important awareness which Makaira nigricans fillets could become a hazardous food item in causing histamine poisoning even though no quality deficiency was observed on the fillets. [source] Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance CharactersJOURNAL OF PHYTOPATHOLOGY, Issue 9 2010Ying Xu Abstract Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty-four single-colony isolates of Xanthomonas oryzae pv. oryzae and 827 single-colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin-sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae. [source] MTHFR C677T Polymorphism and Risk of HCC in Patients With Liver Cirrhosis: Role of Male Gender and Alcohol ConsumptionALCOHOLISM, Issue 1 2009Carlo Fabris Background:, A single nucleotide polymorphism (SNP) C677T in the methylenetetrahydrofolate reductase (MTHFR) gene has been identified. The TT or CT genotypes show a marked reduction of the enzyme activity; this causes higher homocysteine levels and alterations of folate metabolism. Folate metabolism is essential for DNA synthesis and methylation, crucial steps in carcinogenesis. In this paper, we investigated whether the MTHFR C677T SNP could influence the occurrence of hepatocellular carcinoma (HCC) in a cohort of patients transplanted for end stage liver disease of different etiologies. Methods:, Two hundred and twelve consecutive patients who underwent liver transplantation for end stage liver disease due to hepatitis B or C, alcoholic liver disease, and other causes were studied. Two hundred and thirty-six blood donors served as controls. Focal hepatic lesions were searched in the sectioned explanted livers. The presence of the MTHFR C677T SNP was determined via polymerase chain reaction amplification. Results:, Among the 65 patients with HCC, 22 had the CC genotype, 30 the CT, and 13 the TT genotype. Only in patients with alcoholic liver disease was a significant association detected between the TT genotype and the presence of liver cancer (6/17 vs. 5/46, p < 0.05). At stepwise logistic regression analysis the independent selected predictors of HCC were found: age at transplantation >55 years (p < 0.001) and the association among male gender, alcoholic liver disease, and MTHFR TT genotype (p = 0.002). Conclusions:, The present study suggests that male TT carriers with alcoholic cirrhosis bear an increased risk of developing HCC. [source] The role of the gut flora in health and disease, and its modification as therapyALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 8 2002A. L. Hart Summary The gut flora is a vast interior ecosystem whose nature is only beginning to be unravelled, due to the emergence of sophisticated molecular tools. Techniques such as 16S ribosomal RNA analysis, polymerase chain reaction amplification and the use of DNA microarrays now facilitate rapid identification and characterization of species resistant to conventional culture and possibly unknown species. Life-long cross-talk between the host and the gut flora determines whether health is maintained or disease intervenes. An understanding of these bacteria,bacteria and bacteria,host immune and epithelial cell interactions is likely to lead to a greater insight into disease pathogenesis. Studies of single organism,epithelial interactions have revealed the large range of metabolic processes that gut bacteria may influence. In inflammatory bowel diseases, bacteria drive the inflammatory process, and genetic predisposition to disease identified to date, such as the recently described NOD2/CARD15 gene variants, may relate to altered bacterial recognition. Extra-intestinal disorders, such as atopy and arthritis, may also have an altered gut milieu as their basis. Clinical evidence is emerging that the modification of this internal environment, using either antibiotics or probiotic bacteria, is beneficial in preventing and treating disease. This natural and apparently safe approach holds great appeal. [source] INVITED REVIEW: Molecular analysis of predation: a review of best practice for DNA-based approachesMOLECULAR ECOLOGY, Issue 4 2008R. A. KING Abstract Molecular analysis of predation, through polymerase chain reaction amplification of prey remains within the faeces or digestive systems of predators, is a rapidly growing field, impeded by a lack of readily accessible advice on best practice. Here, we review the techniques used to date and provide guidelines accessible to those new to this field or from a different molecular biology background. Optimization begins with field collection, sample preservation, predator dissection and DNA extraction techniques, all designed to ensure good quality, uncontaminated DNA from semidigested samples. The advantages of nuclear vs. mitochondrial DNA as primer targets are reviewed, along with choice of genes and advice on primer design to maximize specificity and detection periods following ingestion of the prey by the predators. Primer and assay optimization are discussed, including cross-amplification tests and calibratory feeding experiments. Once primers have been made, the screening of field samples must guard against (through appropriate controls) cross contamination. Multiplex polymerase chain reactions provide a means of screening for many different species simultaneously. We discuss visualization of amplicons on gels, with and without incorporation of fluorescent primers. In more specialized areas, we examine the utility of temperature and denaturing gradient gel electrophoresis to examine responses of predators to prey diversity, and review the potential of quantitative polymerase chain reaction systems to quantify predation. Alternative routes by which prey DNA might get into the guts of a predator (scavenging, secondary predation) are highlighted. We look ahead to new technologies, including microarrays and pyrosequencing, which might one day be applied to this field. [source] Universal primer cocktails for fish DNA barcodingMOLECULAR ECOLOGY RESOURCES, Issue 4 2007NATALIA V. IVANOVA Abstract Reliable recovery of the 5, region of the cytochrome c oxidase 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all fish species. In this study, we develop and test primer cocktails with a view towards increasing the efficiency of barcode recovery. Specifically, we evaluate the success of polymerase chain reaction amplification and the quality of resultant sequences using three primer cocktails on DNA extracts from representatives of 94 fish families. Our results show that M13-tailed primer cocktails are more effective than conventional degenerate primers, allowing barcode work on taxonomically diverse samples to be carried out in a high-throughput fashion. [source] A fast and inexpensive DNA extraction/purification protocol for brown macroalgaeMOLECULAR ECOLOGY RESOURCES, Issue 2 2007GALICE HOARAU Abstract Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < ,0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated. [source] Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.MOLECULAR ORAL MICROBIOLOGY, Issue 1 2005C. M. Sedgley Background/aims:, Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. Methods:, Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. Results:, Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n = 23), and response to pheromones in E. faecalis culture filtrate (n = 16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n = 31) but not in Enterococcus faecium (n = 2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. Conclusions:, Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones. [source] Lack of association between NPHS2 gene polymorphisms and sporadic IgA nephropathyNEPHROLOGY, Issue 4 2007JIANHUA MAO SUMMARY: Aim: IgA nephropathy (IgAN) is the most common primary form of glomerulonephritis worldwide. In the present study, the genetic structure of the NPHS2 gene was studied to verify if podocin plays a role in the pathogenesis of IgAN. Methods: Clinical characteristics and DNA samples were collected from 26 Chinese children with sporadic IgAN. A direct sequencing was performed after polymerase chain reaction amplification to all the eight exons of the NPHS2 gene. Results: Three synonymous variants as known polymorphisms (954T,C homozygous, 1038A,G heterozygous and homozygous) were found in 3, 4 and 1 patients, respectively. There was no significant difference in the genotypic and allelic frequencies of 954T > C and 1038A > G polymorphisms between the patients and normal controls. Conclusion: No significant difference in the genotypic and allelic frequencies of the identified 954T > C and 1038A > G polymorphisms between the patients and normal controls was found. [source] Polymorphism of renin-angiotensin system genes in IgA nephropathyNEPHROLOGY, Issue 5 2004KENG-THYE WOO SUMMARY: Background and Aims: Individuals are prone to disease because of certain disease-susceptible genes. The angiotensin I-converting enzyme (ACE) gene insertion/deletion (I/D), the angiotensinogen (AGT) gene, M235T, and the angiotensin II type 1 receptor (ATR) gene, A1166C, polymorphisms have been associated with IgA nephropathy (IgAN) and its progression. Several studies on Caucasians and Japanese patients have reported contradictory results. We determined these polymorphisms in 118 Chinese patients with IgAN and 94 healthy Chinese subjects to assess their clinical impact. Methods: Genotyping was performed with DNA isolated from peripheral leucocytes, polymerase chain reaction amplification of the polymorphic sequence, restriction enzymes digestion, and separation and identification of DNA fragments. Clinical data at renal biopsy and final status on renal function were determined from patients' records. Results: Comparing all IgAN patients with controls, AGT and ATR genotype distributions were similar, whereas there was a significant increase in the ACE DD genotype (P < 0.05). When comparing patients with end-stage renal failure (IgAN-ESRF) and those without (IgAN-nonESRF), there was no difference among the three gene polymorphisms. In contrast, there were significant differences in higher male prevalence (P < 0.05), increased serum creatinine at presentation (P < 0.05), more sclerosis (P < 0.01) and higher tubulointerstitial lesion score (P < 0.001) in the IgAN-ESRF group. Conclusion: Among the ACE, AGT and ATR gene polymorphisms, only the DD genotype may predispose the individual to IgAN in the Chinese population. None are significant for prognosticating ESRF. [source] Sudden death caused by chronic Chagas disease in a non-endemic country: Autopsy reportPATHOLOGY INTERNATIONAL, Issue 3 2010Fumiko Satoh Chagas disease is a tropical disease that is prevalent in Latin America. Described herein is an autopsy case of the sudden death of a 48-year-old Brazilian man who had stayed in Japan for 7 years. The man, who had a history of Chagas disease, collapsed unexpectedly at work. Because the cause of death was unknown, forensic autopsy examination was performed. As gross findings, the heart was dilated and rounded with an increase in size and weight. The esophagus and large intestine were dilated moderately, with extensive interstitial inflammatory infiltration in the cardiac muscle, but no apparent parasite nest was observed in various tissues. On post-mortem laboratory examinations, indirect immunofluorescence antibody test indicated the presence of IgG antibody specific to Trypanosoma cruzi in the serum. Subsequent polymerase chain reaction amplification using DNA extracted from blood yielded the specific product derived from T. cruzi genomic DNA. These examinations indicate that the infection had resulted from the Tripanosoma parasite. The cause of death was judged to be chronic cardiomyopathy caused by Chagas disease. It is important for pathologists to know the possible involvement of chronic Chagas disease in sudden unexpected deaths in the current globalized society of Japan. [source] Application of short tandem repeat of genomic DNA and mitochondrial DNA for identification of mixed-up tissue specimensPATHOLOGY INTERNATIONAL, Issue 1 2000Kenji Sano Deoxyribonucleic acid (DNA) typing methods, short tandem repeat of genomic DNA and mitochondrial DNA with the use of polymerase chain reaction amplification, were applied to formalin-fixed, paraffin-embedded tissues submitted for diagnosis, to identify and sort out mixed-up tissue specimens. These techniques were found to be reliable, reproducible and specific for personal identification, and thus to eliminate the need for further examinations and to prevent unnecessary surgery. [source] Detection of resistance to acetolactate synthase inhibitors in weeds with emphasis on DNA-based techniques: a reviewPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 7 2006Cheryl-Ann L Corbett Abstract Resistance to herbicides inhibiting acetolactate synthase (ALS) has been increasing at a faster rate than in any other herbicide group. The great majority of these cases are due to various single-nucleotide polymorphisms in the ALS gene endowing target site resistance. Many diagnostic techniques have been devised in order to confirm resistance and help producers to adopt the best management strategies. Recent advances in DNA technologies coupled with the knowledge of sequence information have allowed the development of accurate and rapid diagnostic tests. While whole plant-based diagnostic techniques such as seedling bioassays or enzyme-based in vitro bioassays provide accurate results, they tend to be labour- and/or space-intensive and will only respond to the particular herbicides tested, making resolution of cross-resistance patterns more difficult. Successful DNA-based diagnosis of ALS inhibitor resistance has been achieved with three main techniques, (1) restriction fragment length polymorphism, (2) polymerase chain reaction amplification of specific alleles and (3) denaturing high-performance liquid chromatography. All DNA-based techniques are relatively rapid and provide clear identification of the mutations causing resistance. Resistance based on non-target mechanisms is not identified by these DNA-based methods; however, given the prevalence of target site-based ALS inhibitor resistance, this is a minor inconvenience. Copyright © 2006 Society of Chemical Industry [source] Phylogenetic Analysis of Complete rRNA Gene Sequence of Nosema philosamiae Isolated from the Lepidopteran Philosamia cynthia riciniTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2010FENG ZHU ABSTRACT. The microsporidian Nosema philosamiae is a pathogen that infects the eri-silkworm Philosamia cynthia ricini. The complete sequence of rRNA gene (4,314 bp) was obtained by polymerase chain reaction amplification with specific primers and sequencing. The sequence analysis showed that the organization of the rRNA of N. philosamiae was similar to the pattern of Nosema bombycis. Phylogenetic analysis of rRNA gene sequences revealed that N. philosamiae had a close relationship with other Nosema species, confirming that N. philosamiae is correctly assigned to the genus Nosema. [source] Development of an Acanthamoeba -specific Reverse Dot-Blot and the Discovery of a New RibotypeTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2001REBECCA J. CAST ABSTRACT. Acanthamoeba is a genus of free-living amoebae, of which some speeies have been found to cause opportunistic infections in humans. The identification of these amoebae in natural and disease samples is based primarily upon morphological features. While these features are more than adequate for identification to the genus level, they are not useful for species-level identification. This not only leads to difficulty in the diagnosis of infections, but it makes an accurate assessment of the natural distribution of acanthamoebae very difficult to achieve. To improve this situation, a detection method was developed that utilizes both selective polymerase chain reaction amplification and the reverse dot-blot. Oligonucleotides were designed to be specific for the described ribosomal groups (or ribotypes) of Acanthamoeba, as well as one specific for the genus itself. When this method was used to analyze a series of Acanthamoeba cultures from Pakistan, a new ribotype was identified in addition to the detection of the ubiquitously distributed T4 type. [source] Human papillomavirus-associated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinomaBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2008D.M. Prowse Summary Background, Human papillomaviruses (HPVs) are sexually transmitted human carcinogens that may play a role in the oncogenesis of penile cancer. Objectives, To investigate the role of HPV infection and expression of the tumour suppressor protein p16INK4A in the pathogenesis of penile cancer. Methods, By means of polymerase chain reaction amplification and reverse hybridization line probe assay to detect HPV infection, and immunohistochemical staining for p16INK4A and Ki67, we analysed 26 penile squamous cell carcinomas (SCCs) and 20 independent penile lichen sclerosus (LS) lesions from 46 patients. Results, HPV DNA was found in 54% of penile SCCs and 33% of penile LS cases in single and multiple infections. High-risk HPV 16 was the predominant HPV type detected. No relationship between Ki67 expression and HPV infection was observed. Strong immunostaining for p16INK4A correlated with HPV 16/18 infection in both penile LS and penile SCC. In our penile SCC series the cancer margins were also associated with penile LS in 13 of 26 lesions, and HPV was detected in seven of the 13 SCC cases associated with LS and in six of the 11 SCC lesions not involving LS. Conclusions, Our study shows a high prevalence of HPV 16 and p16INK4A expression in penile lesions, consistent with an active role for HPV in interfering with the retinoblastoma pathway. High-risk HPV infection could be involved in the tumorigenic process in 50% of penile cancers, and the use of prophylactic HPV vaccines has the potential to prevent these cancers. [source] IgV gene intraclonal diversification and clonal evolution in B-cell chronic lymphocytic leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2006Davide Bagnara Summary Intraclonal diversification of immunoglobulin (Ig) variable (V) genes was evaluated in leukaemic cells from a B-cell chronic lymphocytic leukaemia (B-CLL) case over a 2-year period at four time points. Intraclonal heterogeneity was analysed by sequencing 305 molecular clones derived from polymerase chain reaction amplification of B-CLL cell IgV heavy (H) and light (C) chain gene rearrangements. Sequences were compared with evaluating intraclonal variation and the nature of somatic mutations. Although IgV intraclonal variation was detected at all time points, its level decreased with time and a parallel emergence of two more represented VHDJH clones was observed. They differed by nine nucleotide substitutions one of which only caused a conservative replacement aminoacid change. In addition, one VLJL rearrangement became more represented over time. Analyses of somatic mutations suggest antigen selection and impairment of negative selection of neoplastic cells. In addition, a genealogical tree representing a model of clonal evolution of the neoplastic cells was created. It is of note that, during the period of study, the patient showed clinical progression of disease. We conclude that antigen stimulation and somatic hypermutation may participate in disease progression through the selection and expansion of neoplastic subclone(s). [source] Intra-tumoral interleukin-6 down-regulation system and genetic mutations of tumor suppressor genes in colorectal carcinomaCANCER, Issue 5 2002Chikao Miki M.D. Abstract BACKGROUND The interleukin (IL)-1-IL-6 network, the most potent cascade of pro-inflammatory cytokines, plays an autocrine role in tumor growth. The IL-1-IL-6 network is down-regulated by a phased cytokine inhibitor IL-1 receptor antagonist (ra) and an anti-inflammatory cytokine IL-10. The current study evaluated this down-regulation system in colorectal carcinoma and its relation to the genetic alteration of tumor suppressor genes. METHODS Seventy-four specimens of primary colorectal carcinoma and normal mucosa were collected to measure tissue concentrations of cytokines. Polymerase chain reaction amplification was performed to investigate the loss of heterozygosity of the microsatellite markers on chromosomes 17p and 18q. RESULTS The IL-1ra/IL-6 ratio in the carcinoma specimens was lower than ratios in adenomas and normal mucosae and decreased with disease progression. The IL-1ra/IL-6 ratio in early cancers tended to be lower than that in adenomas and normal mucosae. However, the tissue concentrations of IL-1, and IL-10 were not associated with any clinicopathologic parameters. The tissue IL-1ra concentration correlated with that of IL-6 only in adenomas and early cancers. Immunohistochemically, IL-1ra and IL-6 were localized in the tumor cytoplasm. A reduced tissue IL-1ra/IL-6 ratio in the carcinomas correlated with poor prognosis and was associated with the loss of heterozygosity of the microsatellite markers on chromosomes 18q. CONCLUSIONS There is an IL-6-IL-1ra network system in colorectal tumors, but this system deteriorates with carcinogenesis and tumor growth. The deterioration of this network system was associated with the allelic loss of a portion of chromosome 18q, reflecting the genetic alteration of tumor suppressor genes. Cancer 2002;94:1584,92. © 2002 American Cancer Society. DOI 10.1002/cncr.10324 [source] A GENETIC VARIANT OF APOLIPOPROTEIN M INCREASES SUSCEPTIBILITY TO CORONARY ARTERY DISEASE IN A CHINESE POPULATIONCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2008Wei-Wei Xu SUMMARY 1High-density lipoprotein (HDL) is widely accepted as a lipoprotein that protects against coronary artery and other atherosclerotic diseases. Recently, a new apolipoprotein encoded by the APOM gene, which plays an important role in affecting the intrinsic properties of HDL, has been reported. Genetic variations exist in the APOM gene, but their significance is presently unclear. The aim of the present study was to elucidate whether the APOM T-855C mutant allele is implicated in coronary artery disease (CAD). 2In the present study, 418 patients with CAD and 372 controls were studied, all of whom were Han Chinese from Jiangsu Province, China. Plasma levels of triglycerides (TG), total cholesterol (TC), HDL,cholesterol (HDL-C) and low-density lipoprotein,cholesterol (LDL-C) were evaluated. Genomic DNA from the whole blood from these subjects was subjected to polymerase chain reaction amplification and restriction enzyme digestion to determine genotype with respect to the APOM T-855C polymorphism. 3The allelic frequencies were in Hardy,Weinberg equilibrium. Plasma HDL levels were significantly lower in subjects with CAD than in control subjects (1.08 ± 0.31 vs 1.25 ± 0.32, respectively; P < 0.001) and the distribution of genotypes and allelic frequencies was significantly different in the two groups (P = 0.013 and 0.005, respectively). Multiple logistic regression analysis after adjustment for age, gender, smoking, body mass index, hypertension and serum glucose showed that, compared with the wild-type TT genotype, carriers of the C allele had an increased risk of CAD (odds ratio = 1.819, 95% confidence interval 1.142,2.898; P = 0.012). 4In conclusion, the results of the present study suggest that the APOM T-855C polymorphism carries an increased risk for CAD in this Chinese population. [source] A novel homozygous mutation in the second transmembrane domain of the gonadotrophin releasing hormone receptor geneCLINICAL ENDOCRINOLOGY, Issue 4 2001D. Söderlund BACKGROUND and OBJECTIVE Mutations in the GnRH receptor (GnRH-R) gene cause hypogonadotrophic hypogonadism. Here, we present the molecular studies of the GnRH-R gene in three families with isolated hypogonadotrophic hypogonadism. PATIENTS Three unrelated families, with at least two members diagnosed with isolated hypogonadotrophic hypogonadism were included. MEASUREMENTS DNA sequencing was performed after polymerase chain reaction amplification of each of the three exons of the gene. RESULTS A novel homozygous missense mutation, at nucleotide 268, turning glutamic acid into lysine, located at the second transmembrane domain of the GnRH-R gene was found in two patients pertaining to one of the families studied. Both parents and an unaffected brother were heterozygous carriers of one mutant allele, an unaffected sister was homozygote wild type. In the other two affected families no mutations were found in the GnRH-R gene. CONCLUSIONS This constitutes the first description of an spontaneous mutation located at the second transmembrane domain (Glu90Lys) of the GnRH-R, indicating that the integrity of glutamic acid at this position is crucial for receptor function. Also this report, complementing others, demonstrates that mutations are distributed throughout the GnRH-R gene and that as in the only other homozygous mutation previously described, affected patients present a complete form of hypogonadotrophic hypogonadism. Due to the fact that apparently consanguinity was present in our affected family, we presume that the mutation derived from a common ancestor, by a founder gene effect. [source] | |||||||||||||||||||||||||